Autophagy is a fundamental mechanism for maintenance of cellular homeostasis through self-digestion. Abnormal autophagy is closely related to many human disorders such as cancer, aging, and neurodegeneration. A deep mechanistic understanding of autophagy is crucial for the development of therapeutic strategies against these diseases. Autophagosome biogenesis is intimately associated with the capture and destruction of macromolecules whose turnover is executed in the autophagic process. It entails a spatiotemporal orchestration of protein-membrane interactions which is not fully understood. Dr. Ge has established a functional assay based on cell-free reconstitution of autophagosome biogenesis, through which he has made a further step towards the understanding of autophagosome biogenesis by identifying the ER-Golgi intermediate compartment as a key membrane source of the autophagosome. In this study, Dr. Ge seeks to extend this functional assay to decipher the underlying molecular actions generating the autophagosome. In Aim 1, Dr. Ge will establish a set of new cell-free assays together with cell-imaging and genetics to investigate the initial step of autophagosome biogenesis by focusing on an autophagic signal-induced membrane mobilization event generating the autophagic membrane precursor. In Aim 2, Dr. Ge will develop a systematic protein fractionation approach to identify novel protein factors in autophagosome biogenesis, as well as to define the functional role of each factor in the protein-membrane network of autophagy. Dr. Ge's long term goal is to understand the molecular nature of autophagy in physiological and pathological settings for the purpose of autophagy-modulating therapy. Training in the mentored phase will prepare Dr. Ge to lead an independent research team using biochemical reconstitution, cell imaging, genetic manipulation, and mass spectrometry to address the fundamental questions of autophagy, as well as to understand the pathogenesis of autophagy-related diseases. Training under this award will include: learning new techniques, such as super-resolution imaging, electron microscopy and quantitative mass spectrometry, acquiring more experience in biochemical reconstitution and fractionation, and expanding knowledge and expertise in autophagy, as well as teaching and writing. Completion of the research and training will greatly facilitate Dr. Ge's transition and success as an independent investigator.